2017 Fiscal Year Annual Research Report
Regulation and the role of serine-threonine protein kinase (PBK/TOPK) in spermatogenesis
| Project/Area Number |
17H06799
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| Research Institution | Kyoto University |
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Principal Investigator |
ゴエル サンディープ 京都大学, 農学研究科, 特定准教授 (80801745)
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| Project Period (FY) |
2017-08-25 – 2019-03-31
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| Keywords | Spermatogenesis / Testis / Protein kinase / Null mutants |
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| Outline of Annual Research Achievements |
Generation of PBK/TOPK mutant mice was the main focus last year. Complete bioinformatic analysis of PBK/TOPK locus was completed. Several single guide RNAs (sgRNAs) were designed for genome editing of PBK/TOPK gene by clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated (Cas9) system. Basically, we designed sgRNAs to cut at two sites, within exon 2 and exon 3 of PBK/TOPK gene. It is expected to mutate both exon 2 and exon 3 and at the same time, we expect to have a deletion mutation between exon 2 to 3. The designed sgRNA were testes in cell culture for its efficiency. The evaluated sgRNAs from the cell culture experiment, alone with Cas9 RNA was electroporated into C57BL/6 embryos. The electroporated embryos were surgically transferred to surrogate mother. All the pups born after the transfer were screened for the desired genetic modification using PCR and sequence analysis. Initially, 4 founder mice were identified. On further analysis, 2 founders with 5 bp and 35 bp deletions respectively were selected for generation of homozygous PBK/TOPK null mice. The founder mice are currently been bred to generate homozygous null mutants. We expect the null mice to be available for analysis by the end of May in this year.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Although we are slightly behind schedule as the generation of PBK/TOPK null mice are still in progress, we are quite confident that we will be able to complete all the proposed analysis of null mice in this fiscal year. As mentioned above, we will have the PBK/TOPK null mice by May end which will give us enough time to complete all remaining experiments.
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| Strategy for Future Research Activity |
Initially, plugging efficiency and litter size of PBK/TOPK null mice will be determined and compared with wild-type mice to evaluate their fertility status. The testis tissues of PBK/TOPK null and wild-type mice will be evaluated for histological evaluation. The sperm from PBK/TOPK null and wild-type mice will be quantified and the percentage of dead and live sperm will be assessed. The motility and viability of sperm will also be determined. The interacting partners of PBK/TOPK will be identified using LC-MS/MS. To further confirm and identification of the interacting partners, the western blotting analysis will be done. Influence of PBK/TOPK on several known cells signaling pathways such as WNT-β-CATENIN-TCF-POU5F1/OCT4 (stem cell-specific); GDNF-DAZL (germ cell-specific), HEDGEHOG (spermatogenesis-specific) and TP53 (apoptosis-specific) will be studied. Our preliminary study has shown that JNK, ERK, and p38 MAPK are downstream substrates of PBK/TOPK in testis. From these preliminary results, we would like to decipher the role of PBK/TOPK in molecular pathway of spermatogenesis. Based on the result of LC-MS/MS analysis, new interacting proteins which play role in spermatogenesis will be studied. Expression of spermatogenic cell-specific proteins i.e. early (SCP3 & UCHL1) and late (TNP1 & PRM2) in the null and wild-type mice will be studied by western blot and immunohistochemical analysis. These results will provide insight into the role of PBK/TOPK in spermatogenesis.
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